MATERIALS AND METHOD FOR ASSAYING FOR METHYLATION OF CpG ISLANDS ASSOCIATED WITH GENES IN THE EVALUATION OF CANCER

ABSTRACT

Provided are methods, reagents, and kits for evaluating cancer, such as prostate cancer, in a subject. Disclosed methods of evaluating cancer include methods of diagnosing cancer, methods of prognosticating cancer and methods of assessing the efficacy of cancer treatment. The methods include assaying a biological sample for methylation of a CpG island associated with specified genes. Provided reagents and kits include primers suitable for amplifying at least a portion of a target CpG islands associated with specified genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 12/983,738, filed Jan. 3, 2011, which is a divisional of U.S.patent application Ser. No. 12/115,674, filed on May 6, 2008, abandoned,which is a continuation of International Patent Application No.PCT/US2006/060685, filed Nov. 8, 2006, designating the United States,which claims the benefit of U.S. Provisional Patent Application No.60/734,577, filed Nov. 8, 2005, which are incorporated by referenceherein in their entireties.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 215,818 bytes Byte ASCII (Text) filenamed “714136SequenceListing.txt,” created on Aug. 22, 2013.

BACKGROUND OF THE INVENTION

Phosphate linked cytosine-guanine (CpG) dinucleotides are statisticallyunderrepresented in the genomes of higher eukaryotes, including mammals.The dinucleotide is reportedly found at only 5-10% of its predictedfrequency. The majority of CpG dinucleotides that do remain in the humangenome are normally located within repetitive sequences that arecharacterized by low gene expression levels and exhibit methylation atthe cytosine residues.

CpG islands, on the other hand, represent genomic sequences that containclusters of CpG dinucleotide. CpG islands may be associated with thepromoter region or 5′ end of coding sequences or may be present withinintrons or in genomic regions that are not known to be associated withcoding sequences. They may be unmethylated or methylated in normaltissues and the methylation pattern may be used to control tissuespecific expression and the expression of imprinted genes. Methylationof CpG islands within promoter regions can result in the downregulationor silencing of the associated gene. An increase in methylation ofnormally unmethylated islands is observed in aging tissues even as theoverall methylcytosine content of the DNA is reduced. The aberrantmethylation pattern is more pronounced in cancer cells with increasedmethylation or hypermethylation detected in various cancer tissues. CpGislands may be methylated to varying densities within the same tissue.Thus, aberrant methylation of cytosines within CpG islands can be aprimary epigenetic event that acts to suppress the expression of genesinvolved in critical cellular processes, such as DNA damage repair,hormone response, cell-cycle control, and tumor-celladhesion/metastasis, leading to tumor initiation, progression andmetastasis (Li et al., Biochim. Biophys. Acta, 1704: 87-102 (2004)). Ithas been proposed that a unique profile of promoter hypermethylationexists for each human cancer in which some gene changes are shared andother gene changes are cancer-type specific (Esteller et al., CancerRes., 61: 3225-3229 (2001)). Given that aberrant methylation representsnew information not normally present in genomic DNA and that aberrantmethylation is a common DNA modification and affects a large number ofgenomic targets, it is feasible to develop diagnostic and prognostictests based on information obtained from multiple target CpGs. Suchtests may be based on CpGs that are aberrantly hypermethylated orhypomethylated in the diseased tissues. They may also be based onchanges in methylation density in CpG islands as long as the changescorrolate with the presence of cancer.

Prostate cancer, for example, which is the most common malignancy andthe second leading cause of death among men in the U.S. (Li et al.(2004), supra), has been found to be associated with the methylation ofCpG islands in the promoters of over 30 genes, in particular the CpGisland of the glutathione S-transferase P1 (GSTP1) gene. GSTP1methylation has been detected in over 50% of DNA recovered from urineand plasma of prostate cancer patients (Goessl et al., Ann. N.Y. Acad.Sci., 945: 51-58 (2001); Cairns et al., Clin. Cancer Res., 7: 2727-2730(2001); Jeronimo et al., Urology, 60: 1131-1135 (2002); and Gonzalgo etal., Clin. Cancer Res., 9: 2673-2677 (2003)). However, if diagnosis ofprostate cancer relied solely on the detection of the methylation of theCpG island in the GSTP1 gene, the theoretical limit of the sensitivityof such a test would only be approximately 90%. GSTP1 is also methylatedin prostatic intraepithelial lesions (PIN) which may lead to a falsepositive diagnosis. Some CpG islands are methylated in prostate cancerand other diseases of the prostate, such as benign prostatic hyperplasia(BPH). They may even exhibit some degree of methylation in normal agingprostates. Such markers may not be suitable individually for prostatecancer diagnosis. Therefore, a panel of markers is required to achievethe sensitivity and specificity needed for a clinical test.

The prostate-specific antigen or PSA test continues to be widely used inthe early detection of prostate cancer. While the PSA test has resultedin the majority of prostate cancer cases being diagnosed in asymptomaticmen (Mettlin et al., Cancer, 83(8): 1679-1684 (1998a); Mettlin et al.,Cancer, 82(2): 249-251 (1998b); Humphrey et al., J. Urol., 155: 816-820(1996); and Grossfeld et al., Epidemiol. Rev., 23(1): 173-180 (2001)),the PSA test suffers from poor specificity, which can be as low as 33%when a PSA cut-off level of 2.6 ng/ml is used (Thompson et al., N. Engl.J. Med., 350: 2239-2246 (2004)), even though the sensitivity can be ashigh as 83%. The poor specificity of the PSA test is a direct result ofincreased secretion of PSA in other diseases of the prostate, such asBPH and prostatitis. Thus, an elevated PSA level indicates the need foradditional screening in the form of needle biopsy. Ultimately, theresults of needle biopsies lead to the diagnoses of prostate cancer.

Over 1 million needle biopsies of prostates are performed each year at acost of about $1,500 each and much discomfort to the patient. However,less than 200,000 of these result in a diagnosis of prostate cancer.Therefore, the majority of needle biopsies are being performedneedlessly.

In view of the above, there is a need for non-invasive methods ofdiagnosing and prognosticating cancer, such as prostate cancer, thatreduce the cost and suffering associated with currently available cancerscreening methods. It is an object of the invention to provide materialsand methods for non-invasive diagnosis and prognosis of cancer, such asprostate cancer. This and other objects and advantages, as well asadditional inventive features, will become apparent from the detaileddescription provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides materials and methods for evaluating cancer.Methods of evaluating can include methods of diagnosing andprognosticating cancer as well as methods of assessing the efficacy ofcancer treatment. Generally, the methods provided involve assaying formethylation of CpG islands associated with specific genes. The inventionalso provides pairs of isolated or purified primers that can be used inthe methods of the invention, for example, to amplify and/or detect themethylation state of the CpG islands associated with specific genes. Theinvention also provides kits comprising one or more pairs of primersuseful in the disclosed methods.

The invention provides methods of diagnosing cancer by assaying for oneor more methylated CpG islands that are indicative of cancer. Generally,the method comprises providing a biological sample from a subject inneed of cancer diagnosis and assaying the sample for methylation of oneor more CpG islands associated with at least one gene selected from thegroup consisting of: neuregulin cell-surface ligand (NRG1), adrenergicB3 receptor (ADRB3), glycosylphosphatidyl-inositol cell-surface receptor(GFRA2), kinesin family member 13B (KIF13B), RET proto-oncogene (RET),G-protein-coupled protein receptor 147 (GPR147), neurogenin 3transcription factor (NEUROG3), paladin (predicted protein tyrosinephosphatase) (PALD), methyltransferase family member 1 (HEMK1),fibroblast growth factor 4 oncogene (FGF4), 5-hydroxytryptamine(serotonin) receptor 1A (HTR1A), ring finger protein 180 (LOG 285671 orRNF180), EGFR-co-amplified and overexpressed (DKFZP564K0822 or ECOP),zinc finger protein 596 (ZNF596), similar to 7 transmembrane helixreceptor (LOC441320), L-threonine dehydrogenase (TDH), hypotheticalprotein FLJ36980 (FLJ36980), fibroblast growth factor receptor 20(FGF20), EF-hand domain family member 2A (LOC286097 or EFHA2),N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), nodalhomolog (TGF-β signaling pathway) (NODAL), hypothetical protein similarto zinc finger protein 532 (LOC399783), transcription factor LIMhomeodomain (ISL2) Kinesin family member C2 (KIFC2), chromosome 20 openreading frame 23 (Kinesin-like motor protein) (C20orf23), GDNF familyreceptor alpha 1 (GFRA1), Glutathione peroxidase 7 (GPX7), Dickkopfhomolog 2 (DKK2), netrin 1 (NTN1), matrix metallopeptidase 9 (MMP9),tumor necrosis factor superfamily member 11 (TNFSF11), ras homolog genefamily member D (RHOD), and leucine rich repeat containing 49 (LRRC49).

The invention also provides a method of diagnosing prostate cancer in amale mammal by assaying for one or more methylated CpG islands that areindicative of prostate cancer. The method can include providing abiological sample from a subject in need of cancer diagnosis andassaying the sample for methylation of a CpG island associated with atleast one gene selected from the group consisting of: NRG1, ADRB3,GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4, HTR1A, RNF180,ECOP, ZNF596, LOC441320, TDH, FLJ36980, EFHA2, ASAH1, NODAL, LOC399783,ISL2, MMP9, TNFSF11, RHOD, LRRC49, Kinesin family member C2 (KIFC2),chromosome 20 open reading frame 23 (Kinesin-like motor protein)(C20orf23), GDNF family receptor alpha 1 (GFRA1), Glutathione peroxidase7 (GPX7), Dickkopf homolog 2 (DKK2), netrin 1 (NTN1), Ras association(RalGDS/AF-6) domain family 5 (RASSF5), and HtrA serine peptidase 4(HTRA4). Optionally, the method of diagnosing prostate cancer can alsoinclude assaying for methylation of one or more CpG island associatedwith at least one gene that is known to be methylated in prostate cancerbut is known not to be detectably methylated or is methylated at a lowerlevel (e.g., about 50% or less, about 40% or less, 30% or less, about20% or less, or about 10% or less) in BPH.

The invention also provides methods of prognosticating cancer byassaying for the methylation of one or more genes that are indicative ofthe grade or stage of the cancer, and/or the length of disease-freesurvival following treatment for cancer. Generally, the method comprisesproviding a biological sample from a subject in need of cancer prognosisand assaying the sample for methylation of a CpG island associated withat least one gene selected from the group consisting of: NRG1, ADRB3,GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4, HTR1A, RNF180,DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20, EFHA2, ASAH1,NODAL, LOC399783, ISL2, KIFC2, C20orf23, GFRA1, GPX7, DKK2, NTN1, MMP9,TNFSF11, RHOD and LRRC49.

Further provided by the invention is a method of prognosticatingprostate cancer in a male mammal by assaying for one or more methylatedCpG islands that are indicative of the grade or stage of prostatecancer, and/or the length of disease-free survival following treatmentof prostate cancer. The method comprises providing a biological samplefrom the male mammal and assaying the sample for methylation of a CpGisland associated with at least one of the following genes: NRG1, ADRB3,GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4, GPR62, HTR1A,RNF180, DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20, EFHA2,ASAH1, NODAL, LOC399783, ISL2, KIFC2, C20orf23, GFRA1, GPX7, DKK2, NTN1,RASSF5, HTRA4, MMP9, TNFSF11, RHOD or LRRC49. Optionally, the method ofprognosticating prostate cancer can also include assaying the biologicalsample for methylation of a CpG island associated with at least one genethat is known to be methylated in prostate cancer but is known not to bedetectably methylated or is methylated at a lower level (e.g., about 50%or less, about 40% or less, 30% or less, about 20% or less, or about 10%or less) in BPH. Methylation of the CpG islands associated with thegenes is indicative of the grade or stage of the cancer, and/or thelength of disease-free survival following treatment.

Furthermore, the invention provides methods of assessing the efficacy oftreatment of cancer by assaying for the reduced methylation of CpGislands that indicates efficacy of treatment. Generally, the methodcomprises providing a first and a second biological sample from asubject in need of assessing the efficacy of treatment of cancer andassaying the samples for a change in methylation level of a CpG islandassociated with at least one gene selected from the group consisting of:NRG1, ADRB3, GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4,HTR1A, RNF180, DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20,EFHA2, ASAH1, NODAL, LOC399783, ISL2, KIFC2, C20orf23, GFRA1, GPX7,DKK2, NTN1, MMP9, TNFSF11, RHOD and LRRC49. The first biological sampleis taken before the second biological sample, and the second biologicalsample is taken during or after a course of treatment. A decrease orabsence of methylation of the assayed one or more CpG islands in thesecond sample (i.e., following the course of treatment) indicates thatthe treatment is effective. Alternatively, the maintenance or increaseof methylation in the assayed CpG islands in the second sample canindicate a reduction or absence of treatment efficacy.

Also provided is a method of assessing the efficacy of treatment ofprostate cancer in a male mammal by assaying biological samples, whichare taken from the male mammal periodically during the course oftreatment, for methylation of a CpG island and wherein a decrease orabsence of methylation of the CpG islands following the course oftreatment indicates that the treatment is effective. The methodcomprises (a) providing a first and a second biological sample from asubject undergoing a course of cancer treatment, wherein the firstsample is taken at an earlier time than the second sample, and thesecond sample is taken during or following a course of treatment and (b)assaying the samples for methylation of a CpG island associated with atleast one gene selected from the group consisting of: NRG1, ADRB3,GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4, HTR1A, RNF180,DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20, EFHA2, ASAH1,NODAL, LOC399783, ISL2, KIFC2, C20orf23, GFRA1, GPX7, DKK2, NTN1,RASSF5, HTRA4, MMP9, TNFSF11, RHOD and LRRC49. Optionally, this methodcan also include assaying the biological sample for methylation of a CpGisland associated with at least one gene that is known to be methylatedin prostate cancer but is known not to be detectably methylated or ismethylated at a lower level (e.g. about 50% or less, about 40% or less,30% or less, about 20% or less, or about 10% or less in BPH.

In preferred embodiments, the aforementioned methods of diagnosing,prognosticating and assessing the efficacy of treatment of cancer canfurther include assaying the biological sample for methylation ofmultiple CpG islands, for example, CpG islands associated with two,three, four, five, six, seven, eight, nine, ten, eleven, or more genes.

Additionally, the invention provides a terminator-coupled linearamplification method of determining the methylation status of a CpGisland. Generally, the method includes providing a DNA sample forterminator-coupled linear amplification and then incubating the DNAsample under deaminating conditions to thereby produce a deaminated DNAsample. Optionally, the deaminated DNA sample can be purified. Thedeaminated sample is used as template to amplify a target sequence ortarget sequences that include one or more CpG islands or portions of oneor more CpG islands thereby producing one or more amplified targetsequences. Optionally, the one or more amplified target sequences arepurified. One or more sequences in the amplified target sequences arelinearly amplified in the presence of a primer and a dideoxynucleotideto generate one or more fragments of different lengths, wherein eachlength corresponds to the distance in bases from the 5′ end of theprimer to the position where the dideoxynucleotide is incorporated.Optionally, the one or more fragments is purified. The one or morefragments are analyzed to determine their lengths. The lengths of thefragments can be used to determine the methylation status of methylatedcytosines within the one or more amplified target sequences.

The invention also provides pairs of primers suitable for amplifying aCpG-island associated with genes described herein. Primers can includeisolated or purified nucleic acid molecules suitable for amplifying aCpG island containing target sequence. Target sequences can includegenomic sequence that has been fully methylated and fully deaminatedsuch as those in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54.

Exemplary primer pairs include SEQ ID NOs: 55 and 56, SEQ ID NOs: 57 and58, SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 AND 62, SEQ ID NOs: 63 and 64,SEQ ID NOs: 65 and 66, SEQ ID NOs: 67 and 68, SEQ ID NOs: 69 and 70, SEQID NOs: 71 and 72, SEQ ID NOs: 73 and 74, SEQ ID NOs: 77 and 78, SEQ IDNOs: 79 and 80, SEQ ID NOs: 81 and 82, SEQ ID NOs: 83 and 84, SEQ IDNOs: 87 and 88, SEQ ID NOs: 89 and 90, SEQ ID NOs: 91 and 92, SEQ IDNOs: 93 and 94, SEQ ID NOs: 95 and 96, SEQ ID NOs: 97 and 98, SEQ IDNOs: 103 and 104, SEQ ID NOs: 105 and 106, SEQ ID NOs: 107 and 108, SEQID NOs: 109 and 110, SEQ ID NOs: 111 and 112, SEQ ID NOs: 113 and 114,SEQ ID NOs: 115 and 116, SEQ ID NOs: 117 and 118, SEQ ID NOs: 199 and200, SEQ ID NOs: 201 and 202, SEQ ID NOs: 203 and 204, SEQ ID NOs: 205and 206, SEQ ID NOs: 207 and 208, SEQ ID NOs: 209 and 210, SEQ ID NOs:211 and 212, SEQ ID NOs: 213 and 214, SEQ ID NOs: 215 and 216, SEQ IDNOs: 217 and 218, SEQ ID NOs: 219 and 220, SEQ ID NOs: 221 and 222, SEQID NOs: 224 and 225, SEQ ID NOs: 227 and 228, SEQ ID NOs: 227 and 228,SEQ ID NOs: 230 and 231.

Also provided are kits that include one or more of the aforementionedpairs of primers.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1DD set forth the nucleotide sequences for SEQ ID NOs: 1-54.Sequences are presented in accordance with convention from left to rightand top to bottom.

FIGS. 2A-2KK set forth the nucleotide sequences for SEQ ID NOs: 119-198.Sequences are presented in accordance with convention from left to rightand top to bottom.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of diagnosing cancer by assaying for themethylation of one or more CpG islands that are indicative of cancer.Cancer can include, for example, lung, liver, pancreas, head and neck,throat, thyroid, esophagus, brain, ovarian, kidney, skin, colorectal,and hematopoeietic (e.g., lymphomas and leukemic) cancer. Generally, themethod comprises providing a biological sample from a subject in need ofcancer diagnosis and assaying the sample for methylation of a CpG islandassociated with at least one gene selected from the group consisting of:NRG1, ADRB3, GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4,HTR1A, RNF180, DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20,EFHA2, ASAH1, NODAL, LOC399783, ISL2, KIFC2, C20orf23, GFRA1, GPX7,DKK2, NTN1, MMP9, TNFSF11, RHOD or LRRC49. In preferred embodiments, themethod can include assaying for methylation of CpG islands associatedwith two, three, four, five, six, seven, eight, nine, ten, eleven, ormore of the foregoing genes. Methylation of the CpG islands associatedwith these genes is indicative of cancer.

The invention further provides a method of diagnosing prostate cancer byassaying for the methylation of one or more CpG islands that areindicative of prostate cancer in a male mammal. In one embodiment, themethod comprises providing a biological sample from a male mammal inneed of cancer diagnosis and assaying the sample for methylation of aCpG island associated with at least one gene selected from the groupconsisting of: NRG1, ADRB3, GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD,HEMK1, FGF4, HTR1A, RNF180, DKFZP5640822, ZNF596, LOC441320, TDH,FLJ36980, FGF20, EFHA2, ASAH1, NODAL, LOC399783, KIFC2, C20orf23, GFRA1,GPX7, DKK2, NTN1, HTRA4, MMP9, TNFSF11, RHOD and LRRC49. For example,the method of diagnosing prostate cancer includes assaying thebiological sample for methylation of a CpG island associated with NRG1,KIF13B, or both. In another example, the method includes assaying formethylation of a CpG island associated with at least one gene selectedfrom the group consisting of: TDH, ASAH1, FGF20, HEMK1, PALD NEUROG,EFHA2, KIFC2, GFRA1, DKK2, TNFSF11, NTN1, and RHOD. In preferredembodiments, the method of diagnosing prostate cancer can includeassaying for methylation of CpG islands associated with two, three,four, five, six, seven, eight, nine, ten, eleven, or more of theforegoing genes. Methylation of the CpG islands associated with thesegenes is indicative of cancer.

The foregoing method of diagnosing prostate cancer can optionallyinclude, in combination with assaying for methylation of CpG islandsassociated with the foregoing genes, further assaying the biologicalsample for methylation of a CpG island associated with at least one genethat is known to be (i) methylated in prostate cancer and (ii) notdetectably methylated or methylated at a lower level (e.g., about 50% orless, about 40% or less, about 30% or less, about 20% or less, or lessthan about 10%) in BPH. In this regard, when the method includesassaying for at least one CpG island that is known to be methylated inprostate cancer but is known not to be detectably methylated ormethylated at a lower level in BPH, the method preferably includesassaying the biological sample for methylation of CpG islands associatedwith at least three different genes. Examples of CpG islands known to bemethylated in prostate cancer but not detectably methylated ormethylated at a lower level in BPH include CpG islands associated withglutathione S-transferase P1 (GSTP1), glutathione peroxidase 3 (GPX3),glutathione S-transferase M1 (GSTM1), glutathione S-transferase M4(GSTM4), Cub and Sushi multiple domains1 (CSMD1), tumor necrosis factorreceptor superfamily member 10A (TNFRSF10A) tumor necrosis factorreceptor superfamily member 10B (TNFRSF10B), tumor necrosis factorreceptor superfamily member 10C (TNFRSF10C), tumor necrosis factorreceptor superfamily 10D (TNFRSF10D), secreted frizzled-related protein1 (SFRP1), secreted frizzled-related protein 2 (SFRP2), dickkopf homolog3 (DKK3), prostaglandin-endoperoxide synthase 2 (PTGS2),cyclin-dependent kinase inhibitor 1C (CDKN1C/p57), Ras association(RalGDS/AF-6) domain family 1 (RASSF1), and G-protein coupled receptor62 (GPR62).

The invention also provides a method of prognosticating cancer byassaying for the methylation of one or more genes that are indicative ofthe grade or stage of the cancer, and/or the length of disease-freesurvival following treatment for cancer. Generally, the method comprisesproviding a biological sample from a subject in need of cancer prognosisand assaying the sample for methylation of a CpG island associated withat least one gene selected from the group consisting of: NRG1, ADRB3,GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4, HTR1A, RNF180,DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20, EFHA2, ASAH1,NODAL, LOC399783, ISL2, KIFC2, C20orf23, GFRA1, GPX7, DKK2, NTN1, MMP9,TNFSF11, RHOD and LRRC49. In preferred embodiments, the method caninclude assaying for methylation of CpG islands associated with two,three, four, five, six, seven, eight, nine, ten, eleven, or more of theforegoing genes. Methylation of the CpG islands associated with thesegenes is indicative of the grade or stage of the cancer, and/or thelength of disease-free survival following treatment for cancer.

The invention also provides a method of prognosticating prostate cancerin a male mammal by assaying for the methylation of one or more CpGislands that are indicative of the grade or stage of the prostatecancer, and/or the length of disease-free survival following treatmentfor prostate cancer. In one embodiment, the method comprises assaying abiological sample from the male mammal for methylation of a CpG islandassociated with at least one of the following genes: NRG1, ADRB3, GFRA2,KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4, HTRIA, RNF180,DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20, EFHA2, ASAH1,NODAL, LOC399783, or ISL2. In addition to or instead of the foregoing,the method can include assaying the biological sample for methylation ofa CpG island associated with at least one of the following genes: KIFC2,C20orf23, GFRA1, GPX7, DKK2, NTN1, RASSF5, HTRA4, MMP9, TNFSF11, RHOD orLRRC49. For example, the method of diagnosing prostate cancer includesassaying the biological sample for methylation of a CpG islandassociated with NRG1, KIF13B, or both. In another example, the methodincludes assaying for at least one of the following genes: TDH, ASAH1,FGF20, HEMK1, PALD NEUROG, EFHA2, KIFC2, GFRA1, DKK2, TNFSF11, NTN1, orRHOD. In preferred embodiments, the method of diagnosing prostate caninclude assaying for methylation of CpG islands associated with two,three, four, five, six, seven, eight, nine, ten, eleven, or more of theforegoing genes. Methylation of the CpG islands associated with thesegenes is indicative of the grade or stage of prostate cancer, and/or thelength of disease-free survival following treatment for prostate cancer.

The foregoing method of prognosticating prostate cancer can optionallyinclude, in combination with assaying for methylation of CpG islandsassociated with the foregoing genes, further assaying the biologicalsample for methylation of a CpG island associated with at least one genethat is known to be (i) methylated in prostate cancer and (ii) notdetectably methylated or methylated at a lower level (e.g., about 50% orless, about 40% or less, about 30% or less, about 20% or less, or lessthan about 10%) in BPH. Percent methylation level in BPH refers to thepercent of patients that exhibit some detectable level of methylation atthat locus. In this regard, when the method includes assaying formethylation of at least one CpG island that is known to be methylated inprostate cancer but is known not to be detectably methylated or ismethylated at a lower level in BPH, the method preferably includesassaying the biological sample for methylation of CpG islands associatedwith at least three different genes. Examples of CpG islands known to bemethylated in prostate cancer but not detectably methylated ormethylated at a lower level in BPH include CpG islands associated withGSTP1, GPX3, GSTM1, GSTM4, CSMD1, TNFRSF10A, TNFRSF10B, TNFRSF10C,TNFRSF10D, SFRP1, SFRP2, DKK3, PTGS2, CDKN1C/p57, RASSF1, and GPR62.Methylation of CpG islands associated with the genes is indicative ofthe grade or stage of the prostate cancer, and/or the length ofdisease-free survival following treatment for prostate cancer.

Obtaining information about the aggressiveness of the cancer, its grade,and its stage is helpful when choosing a course of treatment. Thepatterns of CpG methylation may be correlated to the pathological stageand grade of the tumor. For example, in prostate cancer, patterns of CpGmethylation may be correlated to the Gleason score of the primary tumor.The molecular information derived from CpG methylation may also becorrelated to the likelihood of survival and the length of disease-freesurvival following treatment. The above prognostic methods can enablethe prediction of the course of the cancer, as well as the prediction ofthe best approach to treatment.

Also provided are methods of assessing the efficacy of treatment ofcancer by assaying for the reduced methylation of CpG islands thatindicates efficacy of treatment. Generally, the method comprisesproviding a first and a second biological sample from a subject in needof assessing the efficacy of treatment of cancer and assaying thesamples for a change in methylation level of a CpG island associatedwith at least one gene selected from the group consisting of: NRG1,ADRB3, GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4, HTR1A,RNF180, DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20, EFHA2,ASAH1, NODAL, LOC399783, ISL2, KIFC2, C20orf23, GFRA1, GPX7, DKK2, NTN1,MMP9, TNFSF11, RHOD and LRRC49. Generally, the first biological sampleis taken (e.g, prior to commencing treatment or during treatment) beforethe second biological sample, and the second biological sample is takenafter a course of treatment. In preferred embodiments, the methodincludes assaying for a change in methylation of CpG islands associatedwith two, three, four, five, six, seven, eight, nine, ten, eleven, ormore of the foregoing genes. A decrease or absence of methylation of theassayed one or more CpG islands in the second sample (i.e., followingthe course of treatment) indicates that the treatment is effective.Alternatively, the maintenance or increase of methylation in the assayedCpG islands in the second sample can indicate a reduction or absence oftreatment efficacy.

The invention provides a method of assessing the efficacy of treatmentof prostate cancer in a male mammal by assaying for the reducedmethylation of CpG islands that indicate efficacy of treatment ofprostate cancer. In one embodiment, the method comprises assayingbiological samples, which are taken from the male mammal periodicallyduring the course of treatment, for methylation of a CpG islandassociated with at least one gene selected from the group consisting of:NRG1, ADRB3, GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4,HTR1A, RNF180, DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20,EFHA2, ASAH1, NODAL, LOC399783, and ISL2. In addition to or instead ofthe foregoing, the method can include assaying the biological samplesfor methylation of a CpG island associated with at least one geneselected from the group consisting of: KIFC2, C20orf23, GFRA1, GPX7,DKK2, NTN1, RASSF5, HTRA4, MMP9, TNFSF11, RHOD and LRRC49. For example,the method of assessing the efficacy of treatment of prostate cancerincludes assaying the biological sample for methylation of a CpG islandassociated with NRG1, KIF13B, or both. In another example, the methodincludes assaying for a CpG island associated with at least one geneselected from the group consisting of: TDH, ASAH1, FGF20, HEMK1, PALDNEUROG, EFHA2, KIFC2, GFRA1, DKK2, TNFSF11, NTN1, and RHOD. In preferredembodiments, the method can include assaying for methylation of CpGislands associated with two, three, four, five, six, seven, eight, nine,ten, eleven, or more of the foregoing genes. Generally, the assayedbiological samples in the method include a first and a second biologicalsample. The first biological sample can be taken, for example, prior tocommencing treatment or during treatment, though in any event prior totaking the second biological sample. The second biological sample istaken during or after a course of treatment. A decrease or absence ofmethylation of the assayed one or more CpG islands in the second sample(i.e., following the course of treatment) as compared to the firstsample indicates that the treatment is effective. Alternatively, themaintenance or increase of methylation in the assayed CpG islands in thesecond sample as compared to the first sample can indicate a reductionin or absence of treatment efficacy.

The foregoing method of assessing the efficacy of prostate cancertreatment can optionally include, in combination with assaying formethylation of CpG islands associated with the foregoing genes, furtherassaying the biological sample for reduced methylation of a CpG islandassociated with at least one gene that is known to be (i) methylated inprostate cancer and (ii) not detectably methylated or methylated at alower level (e.g., about 50% or less, about 40% or less, about 30% orless, about 20% or less, or less than about 10%) in BPH. In this regard,when the method includes assaying the biological samples for methylationof at least one CpG island that is known to be methylated in prostatecancer but known not to be detectably methylated or methylated at alower level in BPH, the method preferably includes assaying formethylation of CpG islands associated with at least three differentgenes. Examples of CpG islands known not to be methylated in prostatecancer but not detectably methylated or methylated at a lower level inBPH include GSTP1, GPX3, GSTM1, GSTM4, CSMD1, TNFRSF10A, TNFRSF10B,TNFRSF10C, TNFRSF10D, SFRP1, SFRP2, DKK3, PTGS2, CDKN1C/p57, RASSF1, andGPR62. A decrease or absence of methylation of the CpG islandsassociated with the assayed genes in the second sample as compared tothe first sample following some or all of the course of treatmentindicates that the treatment is effective. Alternatively, themaintenance or increase of methylation in the assayed CpG islands in thesecond sample as compared to the first sample can indicate a reductionor absence of treatment efficacy.

CpG islands (Bird, Nature 321: 209-213 (1986); and Gardiner-Garden etal., J. Molec. Biol. 196: 261-282 (1987)) comprise about 1% ofvertebrate genomes and account for about 15% of the total number of CpGdinucleotides. CpG islands typically are between about 0.2 and about 2.0kb in length. They can be located upstream of (e.g., in a promoter orenhancer region) of the coding sequence of the associated genes or theymay also extend into or be found within gene-coding regions of theirassociated genes. A gene-coding region can include exons and introns.Use of the phrase “associated with” to describe a CpG island's relationto a gene, is intended to encompass CpG islands that are upstream ofgene coding sequences as well as internal CpG islands. For example, theCpG island associated with the RET gene is internal and not expected toaffect the expression of the RET gene when methylated. Some CpG islandsare associated with the promoter of two genes and it can affect theexpression of both genes. CpGs were labeled based on their location withrespect to the nearest gene. In some cases, a CpG island may be locatednear the promoter of two different genes and may in this case influencethe expression of both genes. In such case, the CpG island was namedafter one of the genes. For example, the LRRC49 CpG island is alsoassociated with the THAP domain containing 10 (THAP10) gene. A CpGisland can also be associated with a pseudogene or be located in agenomic region that includes no known genes or pseudogenes. The CpGisland can still be of interest so long as its methylation statuscorrelates with a disease status.

A CpG island can be separated by up to 25 kilobases (kb) (e.g., up to 20kb, up to 19 kb, up to 18, kb, up to 17 kb, up to 16 kb, up to 15 kb, upto 10 kb, up to 9 kb, up to 8 kb, up to 7 kb, up to 6 kb, up to 5 kb, upto 4 kb, up to 3 kb, up to 2 kb, or up to 1 kb) from the transcriptionstart site for the nearest gene and still be considered “associatedwith” the gene. Preferably, CpG islands associated with at least threegenes are assayed. However, CpG islands associated with 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, or even more genes can be assayed.

Methods of identifying CpG islands have been described (e.g., Takai etal., Proc. Nat'l. Assoc. Sci. USA, 99:3740-3745 (2002)). For example,genomic sequences can be analyzed to identify segments containing CpGislands that are at least 200 bp in length, have at least a 60% GCcontent, and contain at least 7% CpG dinucleotides. Preferred sequencesare at least 250 bp in length, are at least 60% GC rich, and contain atleast 7% CpG dinucleotides. Moreover, undesirable highly repetitivesequences can be screened out using a repeat masker that filters outsequences. Desirable sequences contain less than 50% repeats (i.e., asequence of reduced complexity or a sequence that is present at multiplegenomic locations) within the length of the identified CpG island.Preferably, the CpG island is no more than 45%, 40%, 35%, 30%, 25%, 20%,19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, or 11% repetitive. Mostdesirable sequences are no more than 10% repetitive. Examples ofrepetitive sequences are available, for example, at the web site forNational Center for Biotechnology Information (NCBI).

“Biological sample” is intended to encompass any suitable sample thatenables accurate assay of CpG island methylation. Examples of suitablebiological samples include, but are not limited to, whole blood, bloodplasma, blood serum, urine, saliva, cells (e.g., cells obtained fromblood, such as epithelial cells), and tissue. Such samples are obtainedin accordance with methods known in the art. When the biological sampleis whole blood, blood plasma, or urine, preferably, CpG islandsassociated with more than three genes are assayed.

A CpG island is “not detectably methylated” when it is not methylated orit is methylated at a level below the level of sensitivity of the assaymethod employed.

“Noncancerous” tissue can be benign or normal. Alternatively, but notpreferably, the tissue can be diseased, as long as it is not cancerous.

Methods of assaying methylation of CpG islands are known in the art andinclude, for example, restriction enzyme-based technology, such as onethat employs digestion with a methylation-sensitive restrictionendonuclease coupled with Southern blot analysis, methylation-sensitiveenzymes and polymerase chain reaction (PCR), such asmethylation-sensitive arbitrarily primed PCR (AP-PCR; see, e.g.,Gonzalgo et al., Cancer Res., 57: 594-599 (1997)), restriction landmarkgenomic scanning (RLGS; see, e.g., Plass et al., Genomic 58: 254-262(1999)), methylated CpG island amplification (MCA; see, e.g., Toyota etal., Cancer Res., 59: 2307-2312 (1999)), differential methylationhybridization (DMH; see, e.g., Huang et al., Human Mol. Genet., 8:459-470 (1999)), and Not I-based differential methylation hybridization(see, e.g., International Patent Publication No. WO 02/086163). Othermethods are described in U.S. Pat. App. Pub. No. 2003/0170684 andInternational Patent Publication No. WO 04/05122.

Alternatively, cytosine conversion-based technology can be used. Suchtechnology relies on methylation status-dependent chemical modificationof CpG islands (i.e., deamination of unmethylated cytosines in CpGislands) within isolated genomic DNA or fragments thereof followed byDNA sequence analysis. Such methods employ reagents like hydrazine andbisulfite. Bisulfite treatment followed by alkaline hydrolysis isdescribed by Olek et al., Nucl. Acids Res., 24: 5064-5066 (1996); andFrommer et al., PNAS USA, 89: 1827-1831 (1992). The use ofmethylation-sensitive primers to assay methylation of CpG islands inisolated genomic DNA is described by Herman et al., PNAS USA, 93:9821-9826 (1996), and in U.S. Pat. Nos. 5,786,146 and 6,265,171.Bisulfite-treated DNA can be subsequently analyzed by conventionalmolecular techniques, such as PCR amplification, fluorescence-based,real-time PCR (see, e.g., Eads et al., Cancer Res., 59: 2302-2306(1999); Heid et al., Genome Res., 6: 986-994 (1996); and U.S. Pat. No.6,331,393), sequencing, oligonucleotide hybridization detection, andmethylation-sensitive single nucleotide primer extension (Ms-SNuPE; see,e.g., Gonzalgo et al., Nucl. Acids Res., 25: 2529-2531 (1997); and U.S.Pat. No. 6,251,594).

A preferred method of assaying for methylation of a CpG island includesisolating genomic DNA (and/or fragments thereof) from a biologicalsample, treating the DNA under deaminating conditions that convertunmethylated cytosines to uracil, using the treated DNA as a template ina PCR reaction to amplify a target sequence that includes the CpG-islandof interest, thereby producing an amplified sequence. Unmethylatedcytosines in the target sequence, which are converted to uracils by thedeaminating treatment, are amplified as thymines in the correspondingposition of the amplified sequence. Since the sequence of the forwardand the reverse strand of the CpG island lose their complimentarityafter the deamination reaction, the methylation status of the CpG islandcan be determined by assaying one or both of the original strands byutilizing primers capable of annealing to the strand of interest.

The deamination reaction may not proceed to completion, which results infalse positives. For example, deamination of DNA sequences usingbisulfite salt is sensitive to the purity of the DNA, length ofincubation, and the secondary structure of the denatured templates.Quantitative PCR methods can be used to assay for the efficiency ofdeamination. However, quantitative PCR methods are limited to assayingthe conversion status within the sites where the primers and probesanneal to the template.

Quantitative PCR methods are also limited to assaying for themethylation of cytosines within the sites where the primers and probesanneal to the template. The primers and the probe only annealefficiently to the templates that are fully converted and containmethylation at the appropriate cytosine nucleotides. Thus, they fail toprovide methylation information for CpG dinucleotides that are notassayed for. The CpG islands may also be analyzed using directsequencing following the deamination treatment. However, due to theheterogeneity of the methylation pattern within a CpG island and thepresence of homopolymeric stretches within the sequence, directsequencing of CpG islands can yield a sequencing pattern that is toonoisy and complex for the available sequencing software.

To overcome these disadvantages and to minimize the overall cost ofanalysis for a clinical test, we developed a method to analyze theamplified sequences by termination-coupled linear amplification. The DNAis linearly amplified using a forward or a reverse primer in thepresence of dNTPs and one or two dideoxynucleotides such asdideoxycytidine or dideoxyguanine. The amplified sequence can,optionally, be analyzed using only thymine and/or cytosine terminatorswhen assaying for a methylated CpG dinucleotide (or adenine and/orguanine terminators when analyzing the amplified strand opposite to theCpG dinucleotide of interest) to make extension reaction products thatterminate at thymines and/or cytosines nucleotides (or at guanine and/oradenine when assaying the opposite strand). The amplification reactionresults in the generation of fragments with multiple lengths, eachlength of which corresponds to the distance in bases between the primerused for amplification and the position within the target sequence of anucleotide that is complementary to the dideoxynucleotide added to theamplification reaction. Such amplification can result in the generationof 10 to 20 fragments from an average CpG island-containing amplicon of100 to 150 bp. The extension products can be separated by size on anacrylamide gel and compared to (a) a size standard and/or (b) bycomparing the fragments to those generated when fully unmethylated (PCRgenerated template or clones in E. coli) or fully methylated(enzymatically methylated in vitro) template to thereby determine thepresence of cytosine (or guanine on the opposite strand) or the presenceof thymine (or adenine on the opposite strand) in the amplified CpGisland-containing sequence. When bisulfite is used as the deaminatingagent, the amplified sequence may contain large stretches of thymine oradenine which may result in additional fragments due to the DNApolymerase slippage during amplification. Such “stutter” patterns may beminimized by selectively analyzing segments of the CpG islands that haveshorter homopolymeric sequences. Stutter fragments can also beidentified by analyzing the control templates.

When a fluoresent label is used to tag the primers or thedideoxynucleotides used in the terminator-coupled linear amplification,the resulting fragments may be analyzed using automated sequencingmachines and software designed for determining the size of DNAfragments. In this regard, commercially available software such asGENESCAN (Applied Biosystems, Foster City, Calif.) and GENEMAPPER(Applied Biosystems) are trained to recognize and account for stutterpatterns due to DNA polymerase slippage during the amplification ofmicrosatellite repeats. Such software may also be used to account forthe stutter pattern that is observed when amplifying homopolymericstretches of DNA, as might be seen after bisulfite conversion of CpGislands. There are a number of fluorescent dyes available for theautomated analysis of DNA such as but not limited to6-carboxyfluorescein (6-FAM), Hexachlorofluorescein (HEX), VIC dye,5-carboxytetramethylrhodamine (TAMRA), 5-carboxy-X-rhodamine,succinimidyl ester (5-ROX), 6-carboxy-2′,4,7,7′-tetrachlorofluorescein(TET). The methods and equipment to determine amplicon size have beenavailable for over a decade and in use for genetic linkage mapping, DNAidentity, and forensic. For example, Applied Biosystems has a set of 5dyes that can be used to multiplex fragments from 4 separateamplification reaction and one standard for use in linkage mapping onthe ABI sequencers. Four different CpG islands from a single individualcan be linearly amplified using fluorescently tagged primers, and theproducts pooled before analysis. Alternatively, different CpG islandsfrom different individuals can be linearly amplified using fluorescentlytagged primers, and the products pooled before analysis.

Since methylation of a particular CpG dinucleotide is not alwayscomplete in a sample, i.e., the CpG sequence is heterogenous, themethods provided herein can be advantageously used to analyze the extentof or percent methylation of a particular CpG dinucleotide site within asample. In a preferred method, two different fluorescent-dye terminatorsare used for thymine and cytosine, respectively (or adenine and guanine,respectively, when analyzing the opposite strand) in a fluorescentdideoxy sequencing reaction. The relative abundance of the two dyes insame-size extension products are indicative of the relative abundance ofthe two nucleotides at a particular sequence position, and can therebyindicate the percent methylation of a particular CpG dinucleotide sitewithin a CpG island. To determine the expected relative abundance of thetwo dyes, control reactions with a range of known ratios of fullymethylated to fully unmethlylated templates can be used. The dataobtained from the control reactions can be used as a reference toestimate relative abundance of methylated and unmethylated cytosines ina sample.

The levels of methylation or patterns of methylation at given CpGislands can be assayed as appropriate. The assay can employ the use of areference standard when appropriate to enable the determination ofabnormal methylation. A reference standard can be determined based onreference samples obtained from age-matched noncancerous classes ofadjacent tissues, and with normal peripheral blood lymphocytes. When,for example, efficacy of treatment is being assessed, the assay resultsof biological samples taken over the course of treatment can be comparedwithout the use of a reference standard.

When the DNA obtained from a biological sample is in limited quantitiesand is not sufficient for the analysis of multiple markers, the methodsdescribed herein can include amplifying the DNA from the sample.Amplification can be done using PCR amplification or isothermalamplification methods, for example, those described in U.S. Pat. Nos.5,854,033; 6,124,120; 6,143,495; 6,210,884; 6,642,034; 6,280,949;6,632,609; and 6,642,034; and U.S. Pat. App. Pub. Nos. 2003/0032024;2003/0143536; 2003/0235849; 2004/0063144; and 2004/0265897, which areincorporated herein by reference in their entirety. Isothermalamplification can include rolling circle or strand displacementamplification. Methods that combine PCR and isothermal amplificationhave also been described (U.S. Pat. Nos. 6,777,187; and 6,828,098; andU.S. Pat. App. Pub. Nos. 2004/0209298; 2005/0032104; and 2006/0068394,each of which is incorporated herein by reference in its entirety). U.S.Pat. App. Pub. No. 2005/0202490, which is incorporated herein byreference in its entirety, describes the use of such methods incombination with methylation-sensitive restriction enzymes to study themethylation pattern of DNA. DNA amplification can also includemethylation-coupled whole genomic amplification to generate the DNAneeded, such as described in U.S. Pat. App. Pub. No. 2006/0257905, whichis incorporated by reference herein in its entirety. Themethylation-coupled whole genomic amplification can be especiallyadvantageous when DNA is recovered from minute biological samples orfrom bodily fluids such as urine or plasma.

Skilled artisans will appreciate that the various amplification methodsdescribed herein, e.g., the PCR amplification, isothermal amplification,and termination-coupled linear amplification method, can employnucleotides, nucleotide analogues, nucleotide or nucleotide analoguederivatives, and/or combinations thereof.

If desired, mRNA and protein levels can be assayed, and alterations intheir expression levels can be indicative of a change in the level ofmethylation or the patterns of methylation at given CpG islands. Suchmethods of assaying mRNA and protein levels are also within the skill inthe art. For example, the mRNA assay methods described in U.S.Provisional Patent Application No. 60/705,964 filed on Aug. 5, 2005 andInternational Patent Publication No. WO 2007/019444, which are herebyincorporated by reference, can be used. Such methods are particularlyuseful if a degraded tissue sample is used as the biological sample.Alternatively, reverse transcription with gene-specific primers can beused to assay mRNA levels. Proteins levels can be assayed, for example,using antibody and staining techniques.

It is important to note that even though aberrant methylation of a CpGisland can affect expression of the associated gene, the methodsdescribed herein are not dependent on a biological role for thehypermethylation. That is a hypermethylated CpG island can be useful inthe methods of the invention regardless of its effect on geneexpression. Accordingly, the only requirement is that there be acorrelation between the methylated state of a CpG island and thepresence of cancer.

The invention further provides target sequences and correspondingprimers or probes that are useful in the above methods. The targetsequences provide the context for the selection of CpG islands to assayfor methylation. If a given target sequence contains more than one CpGisland, all or less than all of the CpG islands, even one CpGdinucleotide, can be assayed for methylation with respect to thatparticular target sequence. In this regard, a target sequence caninclude a genomic sequence that is fully methylated and fully deaminatedsuch as SEQ ID NO: 1 or 2 [NRG1], SEQ ID NO: 3 or 4 [ADRB3], SEQ ID NO:5 or 6 [GFRA2], SEQ ID NO: 7 or 8 [KIF13B], SEQ ID NO: 9 or 10 [RET],SEQ ID NO: 11 or 12 [GPR147], SEQ ID NO: 13 or 14 [NEUROG3], SEQ ID NO:15 or 16 [PALD], SEQ ID NO: 17 or 18 [HEMK1], SEQ ID NO: 19 or 20[FGF4], SEQ ID NO: 23 or 24 [HTR1A], SEQ ID NO: 25 or 26 [RNF180], SEQID NO: 27 or 28 [ECOP], SEQ ID NO: 29 or 30 [ZNF596], ID NO: 33 or 34[LOC441320], SEQ ID NO: 35 or 36 [TDH], SEQ ID NO: 37 or 38 [FLJ36980],SEQ ID NO: 39 or 40 [FGF20], SEQ ID NO: 41 or 42 [EFHA2], SEQ ID NO: 43or 44 [ASAH1], SEQ ID NO: 45 or 46 SEQ ID NO: 49 or 50 [NODAL], SEQ IDNO: 51 or 52 [LOC399783], SEQ ID NO: 53 or 54 [ISL2]. These fullymethylated and deaminated sequences are used for illustrative purposedand do not exclude the use of partially methylated and deaminatedsequences in the methods of the invention. A target sequence can includea genomic sequence that is partially methylated, such as in DNA obtainedfrom a tumor, and then deaminated such that the target differs from thesequence listed above. Persons of skill in the art will appreciate thata target sequence that includes a partially methylated and deaminatedCpG island will result in a population of DNA molecules that differ atone or more positions that correspond to the cytosine residues in one ormore CpG dinucleotides. Thus, a target sequence can include a variety ofpartially methylated and deaminated sequences based on the followinggenomic sequences SEQ ID NOs: 119 or 220 [KIFC2], SEQ ID NOs: 121 or 122[C20ORF23], SEQ ID NOs: 123 or 124 [GFRA1], SEQ ID NOs: 129 or 130[DKK2], SEQ ID NOs: 133 or 134 [RASSF5], SEQ ID NOs: 135 or 136 [NTN1],SEQ ID NOs: 139 or 140 [GPR147], SEQ ID NOs: 141 or 142 [NEUROG3], SEQID NOs: 143 or 144 [NODAL], SEQ ID NOs: 145 or 146 [PALD], SEQ ID NOs:147 or 148 [LOC399783], SEQ ID NOs: 151 or 152 [LOC441320], SEQ ID NOs:153 or 154 [ZNF596], SEQ ID NOs: 155 or 156 [TDH], SEQ ID NOs: 157 or158 [ASAH1], SEQ ID NOs: 159 or 160 [FGF20], SEQ ID NOs: 161 or 162[FLJ36980], SEQ ID NOs: 163 or 164 [GFRA2], SEQ ID NOs: 165 or 166[EFHA2], SEQ ID NOs: 171 or 172 [KIF13B], SEQ ID NOs: 173 or 174[ADRB3], SEQ ID NOs: 175 or 176 [NRG1], SEQ ID NOs: 177 or 178 [ECOP],SEQ ID NOs: 179 or 180 [HTR1A], SEQ ID NOs: 181 or 182 [ISL2], SEQ IDNOs: 183 or 184 [LOC285671], SEQ ID NOs: 185 or 186 [FGF4], SEQ ID NOs:189 or 190, [HEMK1], SEQ ID NOs: 191 or 192 [RET] SEQ ID NOs: 193 or 194[HTRA4], SEQ ID NO: 195 [RHOD], SEQ ID NO: 196[TNFSF11], SEQ ID NO: 197[MMP9], and SEQ ID NO: 198 [LRRC49].

These targets can be used in combination with known targets (for exampleknown CpG islands associated with GSTP1, GPX3, GSTM1, GSTM4, CSMD1,TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, SFRP1, SFRP2, DKK3, PTGS2,CDKN1C/p57, RASSF1, and GPR62. For example, fully methylated anddeaminated sequences for some of these genes are provided in SEQ ID NO:31 or 32 [CSMD1], SEQ ID NO: 45 or 46 [TNFRSF10C], SEQ ID NO: 47 or 48[TNFRSF10B] SEQ ID NO: 21 and 22 [GPR62]. Also for example, a targetsequence can include fully or partially methylated and (subsequently)deaminated sequences based on the following genomic sequences SEQ IDNOs: 131 or 132 [GPX3], SEQ ID NOs: 125 or 126 [GPX7], SEQ ID NOs: 127or 128 [GSTM4], SEQ ID NOs: 137 or 138 [SFRP2], SEQ ID NOs: 149 or 150[CSMD1], SEQ ID NOs: 167 or 168 [TNFRSF10B], SEQ ID NOs: 169 or 170[TNFRSF10C], and SEQ ID NOs: 187 or 188 [GPR62]. Such target sequencescan be isolated or purified in accordance with methods known in the art.

Also provided are isolated or purified primers derived from and suitablefor amplifying sequences internal to the above isolated or purifiednucleic acid molecules. The isolated or purified primers can be DNA,RNA, PNA, and the like. It will be understood by one of ordinary skillin the art, however, that one type of nucleic acid can be preferred overanother, depending on the particular biological sample, the methodologyemployed in assaying CpG islands for methylation, and the ability of theparticular type of nucleic acid to detect methylation. One or more(e.g., two, three four, four, five, six, seven, eight, nine ten or more)isolated pairs of primers can be provided. Optionally, primers areprovided as part of a kit useful in the methods disclosed herein. Thepair of primers can consist essentially of SEQ ID NOs: 55 and 56, SEQ IDNOs: 57 and 58, SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 AND 62, SEQ IDNOs: 63 and 64, SEQ ID NOs: 65 and 66, SEQ ID NOs: 67 and 68, SEQ IDNOs: 69 and 70, SEQ ID NOs: 71 and 72, SEQ ID NOs: 73 and 74, SEQ IDNOs: 75 and 76, SEQ ID NOs: 77 and 78, SEQ ID NOs: 79 and 80, SEQ IDNOs: 81 and 82, SEQ ID NOs: 83 and 84, SEQ ID NOs: 85 and 86, SEQ IDNOs: 87 and 88, SEQ ID NOs: 89 and 90, SEQ ID NOs: 91 and 92, SEQ IDNOs: 93 and 94, SEQ ID NOs: 95 and 96, SEQ ID NOs: 97 and 98, SEQ IDNOs: 99 and 100, SEQ ID NOs: 101 and 102, SEQ ID NOs: 103 and 104, SEQID NOs: 105 and 106, or SEQ ID NOs: 107 and 108. It is understood thatthese primer pairs are examples of suitable primers for use in thecontext of the invention. For example, each primer can be between 10 and40 nucleotides and together the pair of primers can flank a region of atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 150, 200,250, 300 by in length that includes one or more CpG dinucloetides in aCpG island of interest. Primer pairs can be modified in various ways,such as by chemical modification of a base, and still be useful in thecontext of the invention. Other primers derived from the targetsequences, namely SEQ ID NOs: 1-54 and 119-198, and variants thereof,also can be used in the context of the invention. The only requirementis that such primers function to assay for methylation of a given CpGisland. Thus, for example, alternate primers can be selected or theprovided primers can be modified or provided in degenerate form toaccount for target sequence polymorphisms within a given population, solong as the primers are still suitable for assaying modification of CpGislands associated with the genes disclosed herein.

Like the target sequences, the primer pairs can be isolated or purifiedin accordance with methods known in the art. Alternatively, they can besynthesized using routine methods.

The primers can be part of a kit. Preferably, the kit comprises at leastthree pairs of primers, wherein each primer pair is specific for a CpGisland associated with a different gene. However, the kit can compriseadditional primer pairs, such as primer pairs for other CpG islandsassociated with the same gene or primer pairs for amplifying CpG islandsassociated with four, five, six, seven, eight, nine, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or even more genes.The kit can further comprise one or more reagents for assaying formethylation of CpG islands, instructions for use, and/or othercomponents as are typically found in kits. For example, the kit cancomprise a buffer suitable for (a) isolating genomic DNA comprising atarget sequence from a biological sample, (b) amplifying a portion ofthe target sequence, and/or (c) deaminating a target sequence. Inembodiments directed to the evaluation of prostate cancer, a kit cancomprise one or more buffers suitable for preparing genomic DNA fromserum and/or urine samples.

EXAMPLES

The following examples serve to illustrate the invention. The examplesare not intended to limit the scope of the invention.

Example 1

This example demonstrates the determination of the methylation status ofmarkers based on methylation-specific PCR amplification.Paraffin-embedded prostate tissues were obtained following radicalprostatectomies. The tissue samples were sectioned into 23 10-micronsections and slide 1, 12, and 23 were stained using hematoxylin andeosin (H&E). Using the H&E slides as guide, the areas corresponding tothe tumor tissues were microdissected from the unstained slides. Theremaining tissues were recovered to use as a normal paired sample.Following deparaffinization using two xylene extractions and two ethanolwashes, the DNA was isolated from the tumor tissue and surroundingnormal tissues using standard proteinase K digest for 5 days at 50° C.,extraction with phenol/chloroform and ethanol precipitation (CurrentProtocols in Molecular Biology, edited by Ausubel, et al.,Wiley-Interscience (New York 1988, revised 1988-2006)). The DNA wasresuspended in TE8 and the quality and quantity of the DNA was assessedby agarose gel electrophoresis using concentration and size standards asreference. Following denaturation in the presence of 0.3 M NaOH, the DNAwas treated with 2.5 M sodium metabisulfite, pH 5.5, in the presence of1 mM hydroquinone at a concentration of 1 μg of DNA/500 μl. The reactionwas incubated in a thermocycler for a total of 8 cycles (95° C. for 5minutes; 55° C. for 115 minutes).

Following bisulfite treatment, the DNA was purified using the QIAEX IIpurification kit (Qiagen, Valencia, Calif.) according to themanufacturer's recommendations and eluted in 50 μl of TE8. Sodiumhydroxide (5.5 μl of 2 N) was added, and the DNA was incubated at RT for15 min. The DNA was then precipitated with 3 volumes of ethanol and 0.3volumes of 5 M NH₄OAC. The DNA was resuspended in 50 μl of TE8 andstored at −20° C.

In order to determine if a specific CpG position is methylated ingenomic DNA isolated from tumor tissue, methylation-specific polymerasechain reaction (PCR) was performed, using primers designed to overlapthe position of the CpG island of interest. All PCR reactions wereperformed in a MASTERCYCLER thermocycler (Eppendorf, Westbury, N.Y.) for42 cycles of 95° C. for 15 seconds, 63° C. for 30 seconds, and 72° C.for 10 seconds. Each reaction was carried out in 30 μl of 1× PLATINUMTaq PCR buffer containing 1.5 mM magnesium chloride, 0.25 mM dNTPS, 12.5pmoles of each primer, and 0.5 units of PLATINUM Taq enzyme (Invitrogen,Carlsbad Calif.). The primers used for each CpG island and the size ofthe product are shown in Table 1, wherein “F” indicates forward primer,“R” indicates reverse primer, “m” indicates methylated, and “u”indicates unmethylated.

TABLE 1 Annealing Product Gene associated temperature sizewith CpG island Primer sequences (° C.) (bp) NRG1 mF:GAGCGGGTAGCGAGAGTTTCGG 63 119 [SEQ ID NO: 55] mR:TAACGACGCGACTACCGAAAACC [SEQ ID NO: 56] ADRB3 mF:GATTAACGTGTTCGTGATTTCGTT 63 102 [SEQ ID NO: 57] mR:CAACGACCAATAACCAATCAACGCC [SEQ ID NO: 58] GFRA2 mF:ATACGTCGGTGAGTTCGGTTTATC 63 101 [SEQ ID NO: 59] mR:ACTCCCGACTCCCTAAACTCCGAA [SEQ ID NO: 60] KIF13b mF:TGAATCGGCGAGGTGAGAGTCG 65 179 [SEQ ID NO: 61] mR:ACCGAACGTCTCAACGCGAAAACG [SEQ ID NO: 62] RET mF:TATCGTTAGCGTCGTGGTGGAGTT 63 120 [SEQ ID NO: 63] mR:CTACACGAACACTAAACCGACCGA [SEQ ID NO: 64] GPR147 mF:TCGGTCGTTACGTTGATCGTTATTC 63 119 [SEQ ID NO: 65] mR:ACCCTACGCATACCCTTCTCGAAC [SEQ ID NO: 66] NEUROG3 mF:GTTTCGAGGAAGTTTCGGGTACGG 63 103 [SEQ ID NO: 67] mR:GATCGTTAACCTTCTTTCGCCGAC [SEQ ID NO: 68] PALD mF: CGAAGTTGGGAGGAGCGAGTT63 115 [SEQ ID NO: 69] mR: AAACATCCGTACTCCTACGACCGA [SEQ ID NO: 70] HEMK[tiF: CGTATTAGTCGTATTCGCGAGCGT 63  99 [SEQ ID NO: 71] mR:CGAAACTACTCGACCCGACCC [SEQ ID NO: 72] FGF4 mF: TAACGGTACGTTGGAGGTCGAGTT63 102 [SEQ ID NO: 73] mR: ACGACCGCCTCCTTAAACTACGCT [SEQ ID NO: 74]GPR62 mF: TATCGTGTATTCGTTGCGGTTAGG 63 120 [SEQ ID NO: 75] mR:AACGATACGAACGACGTACCGAA [SEQ ID NO: 76] HTR1A mF:TACGTGAATAAGAGGACGTTTCGG 63 115 [SEQ ID NO: 77] mR:AACGATCTTCCGAAATACGCCAA [SEQ ID NO: 78] RNF180 mF:TCGTCGAATCGGTATCGTCGTC 63 118 [SEQ ID NO: 79] mR:ACCTATACCACGTCCCGAAACCT [SEQ ID NO: 80] ECOP mF:CGGTTGTAGTTTGTTCGTTCGTTTC 63 108 [SEQ ID NO: 81] mR:CTAACGCCTCATAACTCCTCGCGT [SEQ ID NO: 82] ZNF596 mF:GCGTCGATTTCGGGAGTAGTATCGT 63  96 [SEQ ID NO: 83] mR:ATACCGTAAATCCGCGCTACTTCC [SEQ ID NO: 84] CSMD1 mF:CGTTGAGGTCGAATGAAGCGTAGT 63  96 [SEQ ID NO: 85] mR:AACCGAAACTAAACACGACGCAA [SEQ ID NO: 86] LOC441320 mF:AAGCGTATAGTTCGAGGATTGCGA 63 107 [SEQ ID NO: 87] mR:CCGCGTCACTTACTCCTCACGA [SEQ ID NO: 88] TDH mF: CGTTGGGTGCGTAGGAAGGTTAGT63 120 [SEQ ID NO: 89] mR: GACCGACCCTAAACAACCCGCT [SEQ ID NO: 90]FLJ36980 mF: GTTGCGGGATAGCGTTGTGATT 63  96 [SEQ ID NO: 91] mR:ACCATTATCAATACTCCGATCGCC [SEQ ID NO: 92] FGF20 mF:TTTGTTTGTTAAGGGCGTTATCGT 63 105 [SEQ ID NO: 93] mR:CCGCGACTACTCTAACCAACCC [SEQ ID NO: 94] EFHA2 mF: GGGCGTTGAGTTTAGTTCGGAGA63 108 [SEQ ID NO: 95] mR: ACGAACACAACCGAATCAACGTAA [SEQ ID NO: 96]ASHA1 mF: GGCGTTGGTTGTTAGAGCGATG 63 114 [SEQ ID NO: 97] mR:GACTCAAACTCACTCACCGACGAC [SEQ ID NO: 98] TNFRSF10C mF:GGTGCGATTTAGGATTTAGGACGG 63 115 [SEQ ID NO: 99] mR:GCGACCGAAACTCACTAACAACAA [SEQ ID NO: 100] TNFRSF10B mF:GCGATTTGGGTCGTTAGGGAATAG 63 119 [SEQ ID NO: 101] mR:ACCTCTCCGTAACTTCACGCAACTT [SEQ ID NO: 102] NODAL mF:GGTAGTCGCGGTCGTTTACGTT 63 111 [SEQ ID NO: 103] mR:ACGAACAAACGACAAATCGAATCA [SEQ ID NO: 104] LOC399783 mF:TACGTTGAGTTCGGTTTGGTTTGT 63 103 [SEQ ID NO: 105] mR:CGCGCCTCCGTAATCTAAACTAA [SEQ ID NO: 106] ISL2 mF:GTGCGTGTTGACGTTATGTTGCGT 63  99 [SEQ ID NO: 107] mR:CGCCCGACCTCGACTCTTTACT [SEQ ID NO: 108] GSTP1 mF:CGGCGATTTCGGGGATTTTAGGGC 63 109 [SEQ ID NO: 109] mR:GACGCTCTTCTAAAAAATCCCGCG [SEQ ID NO: 110] GSTP1 mF:ACGTTCGGGGTGTAGCGGTCGTC 63  93 [SEQ ID NO: 111] mR:CCCCAATACTAAATCACGACGCCG [SEQ ID NO: 112] GSTP1 mF:GGTCGGCGTCGTGATTTAGTATTGG 63  99 [SEQ ID NO: 113] mR:ACTACGACGACGAAACTCCAACGA [SEQ ID NO: 114] GSTP1 uF:TGTGGTGATTTTGGGGATTTTAGGGT 63 113 [SEQ ID NO: 115] uR:CCAACCACTCTTCTAAAAAATCCCACA [SEQ ID NO: 116] GSTP1 uF:GATGTTTGGGGTGTAGTGGTTGTTG 63  99 [SEQ ID NO: 117] uR:CTCCACCCCAATACTAAATCACAACA [SEQ ID NO: 118] KIFC2 mF:TGATGGTCGTATTGCGGGTTTATC 62  91 [SEQ ID NO: 199] mR:ATACCTAAACCCAACGCCGACTAC [SEQ ID NO: 200] C20orf23 mF:CGCGATTTGAGTAGTTAGCGTCGT 62  90 [SEQ ID NO: 201] mR:AACCAACGCGACGACCTAACTAAC [SEQ ID NO: 202] GFRA1 mF:TAGATTTCGGTGTTTCGGGCGTT 62  98 [SEQ ID NO: 203] mR:CCGCTAATTCCCAATCGTACTACTCA [SEQ ID NO: 204] GPX7 mF:TTCGTTTCGTTCGGTCGTGATT 62 116 [SEQ ID NO: 205] mR:GACTACGAACGCTTCGAATTCCTC [SEQ ID NO: 206] DKK2 mF:GTTGCGTTGGTAGCGATTCGTTGT 62 117 [SEQ ID NO: 207] mR:CCCGAACCGAATCCTCGAAATCT [SEQ ID NO: 208] NTN1 mF:GACGTAGTATGATGCGCGTAGTGTG 62 103 [SEQ ID NO: 209] mR:GCGAACATACTAAACCCGAACCC [SEQ ID NO: 210] HTRA4 mF:GGATTACGTCGGTGTTCGATTTGT 62  95 [SEQ ID NO: 211] mR:AACGCACGATTAACCCTACGCC [SEQ ID NO: 212] MMP9 mF:TCGGATTAAGGTAGGCGTGGTTTC 62 102 [SEQ ID NO: 213] mR:AACGTAAACGCCGAACCGAAC [SEQ ID NO: 214] RHOD mF: GGAAGACGTCGTTGTTGATGGTTT62 120 [SEQ ID NO: 215] mR: ACCGCTCCGACACGAACCTATAC [SEQ ID NO: 216]TNSF11 mF: AGCGTTATGCGTCGCGTTAGTAG 62 116 [SEQ ID NO: 217] mR:GCAAACGACGACGAAACGTACA [SEQ ID NO: 218] SFRP2 mF:GAAGAGAGCGGGTTCGGGATAAG 62 101 [SEQ ID NO: 219] mR:CTACAACATCGTAAACGCGCGAC [SEQ ID NO: 220]

The products of the PCR reactions were separated on 8% acrylamide gel.Only templates that exhibited methylation at all of the CpG islands thatwere present within the primers could serve as efficient templates forthe amplification reactions. Control reactions were performed usingfully methylated templates that were methylated in vitro using SS1 (CpG)methylase (NEB, Beverly, Mass.) according to the manufacturer'sprotocol. All primer pairs listed in Table 1 yielded a product of thecorrect size from fully methylated control template. Two negativecontrols (water and DNA isolated from white blood cells) were includedfor each target PCR amplification, which did not yield a PCR product.When a CpG island is methylated in a DNA sample, an amplificationproduct of the expected size is obtained. This example demonstrates thatthe above primers can be used to assay for methylation of CpG islands inprostate cancer and that the CpG islands exhibit methylation in prostatecancer.

Example 2

This example demonstrates the determination of the methylation status ofCpG islands at the ADRB3 locus by DNA sequencing. DNA is obtained fromtumor samples and treated with sodium bisulfite as described inexample 1. Two microliters of the bisulfite treated DNA are amplifiedwith the following primers: ADRB3-F1: GAGAAGAGGAAGGTAGAAGGAG [SEQ ID NO:221] and ADRB3-R1: CTACCTAACTATAACCAACCC [SEQ ID NO: 222] for 40 cyclesas described in example 1 except for the annealing temperature, which islowered to 55° C. The amplified 250 bp product is purified usingQIAquick PCR purification kit (Qiagen, Valencia Calif.) and recovered inTE8. Fifty nanograms of the ADRB3 amplified product is sequenced using1.25 pmole of ADRB3-F2:ACGGAGGAGGATAGTAGTACG [SEQ ID NO: 223] usingBigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and thesequencing reaction is purified using Centri-Sep columns (AppliedBiosystems) according to the manufacturer's protocols. The products ofthe sequencing reaction are analyzed using an ABI 3700 sequenceraccording to manufacturer's specification. The resulting DNA sequenceshows one or more sequence peaks corresponding to cytosine base or amixed cytosine/thymidine base at the cytosine residue position of CpGdinucleotides that are fully or partially methylated in the originaltumor DNA.

Alternatively, a more detailed sequence analysis is obtained by cloningthe product of the amplification reaction using a TOPO TA cloning kit(Invitrogen, Carlsbad Calif.) according to supplier's protocol.Approximately 20 colonies are chosen for further analysis. Each colonyis grown in 3 ml of LB media for 16 hours. DNA is isolated from 1.5 mlaliquot using plasmid preparation kit from Qiagen. The plasmid DNA isquantitated using spectrophotometer and 1 microgram aliquot is sequencedas described above. The sequence of the 20 individual clones is comparedto determine which cytosines are methylated and to provide an estimateof their rate of methylation in the tumor sample. This example showsthat the methylation status of cytosines within CpG islands can bedetermined using a sequencing approach.

Example 3

This example demonstrates the determination of the methylation patternof multiple CpG islands associated with KIFC2, GFRA1 and GPX7 usingterminator-coupled linear amplification. From DNA from tumor samplesprepared as described in example 1, fragments of the CpG islandsassociated with KIFC2, GFRA1, GPX7 are amplified individually using themF1 and mR1 primers shown below for each CpG island. The amplificationreactions are performed for 42 cycles as described in example 1 exceptfor the annealing temperature, which was lowered to 58° C. An aliquot ofthe amplification reaction is separated on an 8% acrylamide gel toverify that fragments of the appropriate length are obtained (264 bp forKIFC2, 326 bp for GFRA1, 367 bp for GPX7). The product of the PCRreaction were purified using QIAQUICK PCR purification kit (Qiagen).

Each amplification product (25 nanograms) is subjected to linearterminator-coupled amplification using 1.5 pmoles of the fluorescentlylabeled F2 primer shown below for the corresponding amplicon. Theamplification reaction includes 1× VentR (exo-) DNA polymerase (NewEngland Biolabs, Beverly Mass.), 30 μM dATP, 37 μM dCTP, 100 μM dGTP,100 μM dTTP, 480 μM ddCTP and 2 units of VentR (exo-) DNA polymerase.Reactions are performed in an MASTERCYCLER thermocycler (Eppendorf) for30 cycles of 95° C. for 15 seconds, 58° C. for 30 seconds, and 72° C.for 30 seconds. Following amplification, the reaction products arepooled into a single tube and purified using Centri-Sep columns (AppliedBiosystems) according to the manufacturer's protocols. One microliter ofGENESCAN 500 LIZ standard (Applied Biosystems) is added to one tenth ofthe purified fragment and the DNA separated using the ABI Prism 3100Genetic Analyzer (Applied Biosystems) according to manufacture'sinstructions. The data is analyzed using the GENESCAN and the GENEMAPPERsoftware (Applied Biosystems).

The following primers are used for the amplifications:

KIFC2-F1: [SEQ ID NO: 224] AGGTA(C/T)GTTGTATTTGGTGGATTTGG KIFC2-R1:[SEQ ID NO: 225] CCCACCTACAACAACAACACC KIFC2-F2: [SEQ ID NO: 226]6FAM-GAACGCGTACGGAAGGTAGG GFRA1-F1: [SEQ ID NO: 227]GTGATAGGTTTGTAGATTTGATAGTTG GFRA1-R1: [SEQ ID NO: 228]AACTAACCTCCATTTTAACTATTTC GFRA1-F2: [SEQ ID NO: 229]NED-GAGAGATGAATTTGGATATTAGT GPX7-F1: [SEQ ID NO: 230]GGTAAATTGGTGT(C/T)GTTGGAGAAG GPX7-R1: [SEQ ID NO: 231]ACTAAACAATAATACCC(A/G)ACCTC GPX7-F2: [SEQ ID NO: 232]VIC-GTCGTTGGGTTCGGTTTCGTTTTG

The F1 and R1 primers are used for the amplification of a fragment of aCpG island from the tumor DNA. The F2 primers are used fortermination-coupled linear amplification.

This example shows that termination-coupled linear amplificationfragment lengths can be analyzed to (i) determine the presence and/orthe positions of methylated cytosines in CpG islands in a sequence ofinterest as well as (ii) provide information about the efficiency of thedeamination reaction, since incomplete deamination results in fragmentswith length that differ than what is expected from the positions of theCpG dinucleotides within the sequence.

Example 4

This example demonstrates the use of methylation-coupled whole genomeamplification on DNA recovered from urine samples to increase the amountof DNA available for CpG island marker assays. Urine samples wereobtained from 4 patients that were recently diagnosed with prostatecancer. 50 ml samples were spun down at 4000 rpm for 15 min, transferredto 1.5 ml tubes and washed twice with PBS. The DNA was extracted usingproteinase K digest (100 μl of 25 mM Tris pH8.0, 100 mM NaCL, 1% SDS, 5mM EDTA and 10 μg of Proteinase K followed by phenol/chloroformextraction and ethanol precipitation. The DNA was resuspended in 10 μlTE8 buffer (10 mM Tris, pH 8.0, 1 mM EDTA).

A partially random primer with the sequence GGGN₆ (50 ng) was added to 5μl of DNA. 12 μl of a denaturing solution (50 mM KOH, 0.1 mM EDTA) wasadded to the DNA/random primer mix. After a five-minute incubation atroom temperature, 12 μl of a neutralization solution (60 mM Tris (pH7.5), 50 mM HCl) was added to neutralize the reaction. The DNA/primermix was denatured at 94° C. for 5 minutes, incubated at room temperaturefor 10 minutes, and then placed on ice.

The amplification reaction was set up in a final volume of 30 μl. Thefollowing reagents were added to give the indicated finalconcentrations: (a) 1×NEB buffer 2 (1×NEB buffer 2: 50 mM NaCl, 10 mMTris-HCl, pH 7.9, 10 mM MgCl₂, 1 mM dithiothreitol), 333 μM dATP, dCTP,dGTP, dTTP, 160 μM S-adenosylmethionine, and 10 ng/μl of bovine serumalbumin (BSA) were combined and to which was added (b) DNAmethyltransferase enzyme 1 (0.15 units/μl) (New England Biolabs) andincubated at 37° C. for 10 minutes, and followed by (c) adding Klenowpolymerase to a final concentration of 0.167 units/μl, and Klenow exo-to a final concentration of 0.167 units/p1 (New England Biolabs).

The reaction was incubated at 37° C. for 16 hours, and the reaction wasstopped by the addition of EDTA to a final concentration of 5 mM,phenol/chloroform extracted, and ethanol precipitated. The DNA wasresuspended in 40 μl of TE8 and 2 μl were separated on agarose gel toverify the presence of DNA.

The DNA was treated with sodium bisulfite and analyzed by methylationspecific PCR as described in Example 1 using the GPR147 and RET assays.The presence of a band of the expected size for either marker indicatedthe methylation of the associated marker in the input DNA.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a,” “an,” “the,” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Itshould be understood that the illustrated embodiments are exemplaryonly, and should not be taken as limiting the scope of the invention.

1. A method of determining the methylation status of one or more CpGislands indicative of prostate cancer in a human male undergoingprostate cancer evaluation, which method comprises: (a) isolating oramplifying genomic DNA from a biological sample from a human maleundergoing prostate cancer evaluation; and (b) assaying the genomic DNAfor methylation of one or more CpG islands including a CpG islandassociated with the Ras association (RalGDS/AF-6) domain family 5(RASSF5) gene, wherein the presence of methylation of the one or moreCpG islands, including the CpG island associated with RASSF5, isindicative of prostate cancer.
 2. The method of claim 1, wherein themethod further comprises assaying the isolated or amplified genomic DNAfor methylation of one or more CpG islands associated with nodal homolog(TGF-β signaling pathway) (NODAL), methyltransferase family member 1(HEMK1), glutathione peroxidase 7 (GPX7), paladin (predicted proteintyrosine phosphatase) (PALD), kinesin family member 13B (KIF13B),kinesin family member C2 (KIFC2), or neurogenin 3 transcription factor(NEUROG3), and wherein the presence of methylation of the one or moreCpG islands associated with NODAL, HEMK1, GPX7, PALD, KIF13B, KIFC2, orNEUROG3 is indicative of prostate cancer.
 3. The method of claim 2,wherein the method further comprises assaying the isolated or amplifiedgenomic DNA for methylation of CpG islands associated with NODAL, HEMK1,GPX7, PALD, KIF13B, KIFC2, and NEUROG3, and wherein the presence ofmethylation of the CpG islands is indicative of prostate cancer.
 4. Themethod of claim 2, wherein the method comprises assaying the isolated oramplified genomic DNA for methylation of one or more CpG islands in SEQID NOS: 49 or 50 [NODAL], SEQ ID NO: 17 or 18 [HEMK1], SEQ ID NOs: 125or 126 [GPX7], SEQ ID NO: 15 or 16 [PALD], SEQ ID NOs: 7 or 8 [KIF13B],SEQ ID NOs: 119 or 220 [KIFC2], or SEQ ID NOs: 141 or 142 [NEUROG3], andwherein the presence of methylation of the one or more CpG islandsassociated with NODAL, HEMK1, GPX7, PALD, KIF13B, KIFC2, or NEUROG3 isindicative of prostate cancer.
 5. The method of claim 1, wherein themethod further comprises assaying the isolated or amplified genomic DNAfor methylation of one or more CpG islands associated with at least onegene that is known to be methylated in prostate cancer and that is knownnot to be detectably methylated or methylated at a lower level in benignprostate hyperplasia (BPH), and wherein the presence or increasedmethylation of the assayed CpG islands is indicative of prostate cancer.6. The method of claim 2, wherein the method further comprises assayingthe isolated or amplified genomic DNA for methylation of one or more CpGislands associated with at least one gene that is known to be methylatedin prostate cancer and that is known not to be detectably methylated ormethylated at a lower level in benign prostate hyperplasia (BPH),wherein the presence or increased methylation of the assayed CpG islandsis indicative of prostate cancer.
 7. The method of claim 5, wherein theone or more CpG islands associated with at least one gene that is knownto be methylated in prostate cancer and that is known to be unmethylatedor methylated at a lower level in BPH is or includes one or more CpGislands associated with glutathione S-transferase P1 (GSTP1),glutathione peroxidase 3 (GPX3), cyclin-dependent kinase inhibitor1C(CDKN1C/p57), or G-protein coupled receptor 62 (GPR62).
 8. The methodof claim 7, wherein the one or more CpG islands associated with at leastone gene that is known to be methylated in prostate cancer and that isknown to be unmethylated or methylated at a lower level in BPH includesa CpG island associated with glutathione S-transferase P1 (GSTP1). 9.The method of claim 1, wherein the method further comprises assaying theisolated or amplified genomic DNA for methylation of one or more CpGislands associated with L-threonine dehydrogenase (TDH),N-acylsphingosine amidohydrolase (acid ceraminase) 1 (ASAH1), GDNFfamily receptor alpha 1 (GFRA1), Dickkopf homolog 2 (DKK2), tumornecrosis factor superfamily member 11 (TNFSF11), or leucine rich repeatcontaining 49 (LRRC49), and wherein the presence of methylation of theone or more CpG islands is indicative of prostate cancer.
 10. The methodof claim 5, wherein the method further comprises assaying the isolatedor amplified genomic DNA for methylation of one or more CpG islandsassociated with L-threonine dehydrogenase (TDH), N-acylsphingosineamidohydrolase (acid ceraminase) 1 (ASAH1), GDNF family receptor alpha 1(GFRA1), Dickkopf homolog 2 (DKK2), tumor necrosis factor superfamilymember 11 (TNFSF11), or leucine rich repeat containing 49 (LRRC49), andwherein the presence of methylation of the one or more CpG islands isindicative of prostate cancer.
 11. The method of claim 9, wherein theassaying for methylation of the at least one CpG island associated witha gene comprises amplifying a target sequence that includes at least oneCpG island in one or more of (a), SEQ ID NOs: 35 or 36 [TDH], SEQ IDNOs: 43 or 44 [ASAH1], SEQ ID NOs: 123 or 124 [GFRA1], SEQ ID NOs: 129or 130 [DKK2], SEQ ID NO: 196 [TNFSF11], and SEQ ID NO: 198 [LRRC49],and (b) fully or partially methylated sequences of (a).
 12. The methodof claim 9, wherein the method comprises assaying for methylation of CpGislands associated with at least 7 genes.
 13. The method of claim 9,wherein the method comprises assaying for methylation of CpG islandsassociated with at least 8 genes.
 14. The method of claim 9, wherein themethod comprises assaying for methylation of CpG islands associated withat least 9 genes.
 15. The method of claim 1, wherein the method furthercomprises assaying the isolated or amplified genomic DNA for methylationof one or more CpG islands associated with nodal homolog (TGF-βsignaling pathway) (NODAL), methyltransferase family member 1 (HEMK1),glutathione peroxidase 7 (GPX7), paladin (predicted protein tyrosinephosphatase) (PALD), kinesin family member 13B (KIF13B), kinesin familymember C2 (KIFC2), neurogenin 3 transcription factor (NEUROG3),glutathione S-transferase P1 (GSTP1), glutathione peroxidase 3 (GPX3),cyclin-dependent kinase inhibitor 1C (CDKN1C/p57), or G-protein coupledreceptor 62 (GPR62), L-threonine dehydrogenase (TDH), N-acylsphingosineamidohydrolase (acid ceraminase) 1 (ASAH1), GDNF family receptor alpha 1(GFRA1), Dickkopf homolog 2 (DKK2), tumor necrosis factor superfamilymember 11 (TNFSF11), and leucine rich repeat containing 49 (LRRC49), andwherein the presence of methylation of the one or more CpG islandsassociated with NODAL, HEMK1, GPX7, PALD, KIF13B, KIFC2, NEUROG3, GSTP1,GPX3, CDKN1C/p57, GPR62, TDH, ASAH1, GFRA1, DKK2, TNFSF11, or LRRC49 isindicative of prostate cancer
 16. The method of claim 1, wherein theassaying for methylation of a CpG island comprises amplifying a targetsequence that includes a CpG island in SEQ ID NO: 133 or SEQ ID NO: 134.17. The method of claim 1, wherein assaying the genomic DNA formethylation comprises terminator-coupled linear amplification.
 18. Themethod of claim 1, wherein assaying the genomic DNA for methylationcomprises using methylation-sensitive restriction endonuclease.
 19. Themethod of claim 1, wherein assaying the genomic DNA for methylationcomprises differential methylation hybridization.
 20. The method ofclaim 1, wherein the amplification of genomic DNA comprises methylationcoupled genomic amplification.
 21. The method of claim 1, whereinassaying the genomic DNA for methylation comprises quantitative PCR. 22.The method of claim 1, wherein assaying the genomic DNA for methylationcomprises sequencing.
 23. The method of claim 1, wherein the biologicalsample is whole blood, blood plasma, or blood serum.
 24. The method ofclaim 1, wherein the biological sample is urine.
 25. The method of claim1, wherein the biological sample is prostate tissue.